Little is known about the role of global phosphorylation events in the control of prokaryote metabolism. By performing a detailed analysis of all protein phosphorylation events previously reported in Escherichia coli, dynamic changes in protein phosphorylation were elucidated under three different culture conditions. Using scheduled reaction monitoring, the phosphorylation ratios of 82 peptides corresponding to 71 proteins were quantified to establish whether serine (S), threonine (T) and tyrosine (Y) phosphorylation events displayed a dynamic profile under changing culture conditions. The ratio of phosphorylation for 23 enzymes from central carbon metabolism was found to be dynamic. The data presented contributes to our understanding of the global role of phosphorylation in bacterial metabolism and highlight that phosphorylation is an important, yet poorly understood, regulatory mechanism of metabolism control in bacteria.